CN114853757B - Bifunctional macrocyclic chelant derivatives and methods of making the same - Google Patents

Abstract

The invention belongs to the field of preparation of radiopharmaceuticals, in particular relates to the field of preparation of bifunctional macrocyclic chelant derivatives, and more particularly relates to a bifunctional macrocyclic chelant derivative and a preparation method thereof. Meanwhile, the invention provides a preparation method of the NCS group-containing bifunctional macrocyclic chelant derivative compound II, which is simple in process and suitable for industrial production, and can rapidly and efficiently realize the purification and refining of the compound I through the conversion connection of the purification intermediate compound III.

Description

Bifunctional macrocyclic chelant derivatives and methods of making the same

Technical Field

The invention belongs to the field of preparation of radiopharmaceuticals, in particular relates to the field of preparation of bifunctional macrocyclic chelant derivatives, and more particularly relates to a bifunctional macrocyclic chelant derivative and a preparation method thereof.

Background

Radionuclides typically require attachment to a target molecule via a bifunctional chelator to form a radioactive probe. The stability of a radioactive probe in vivo is an important criterion for judging its performance. Because of the advantages and disadvantages of the radionuclides, respectively, the ideal radionuclide is not present, and thus different nuclides in the prior art need to match different bifunctional chelators.

The currently commonly used cyclic chelating agents are DOTA and NOTA or derivatives thereof, and DOTA can chelate diagnostic nuclides ⁶⁸ Ga(t _1/2 ＝68.1min)、 ¹¹¹ In and therapeutic nuclides ¹⁷⁷ Lu, but DOTA does not chelate Al ¹⁸ F](t _1/2 =109.8 min) and ⁸⁹ Zr(t _1/2 =78.4h), NOTA can be used to chelate Al [ [ ¹⁸ F]、 ⁸⁹ Zr, DOTA and NOTA can only specifically sequester one or several radionuclides and cannot sequester radionuclides in a broad spectrum.

Thus, the preparation and development of a broad spectrum of bifunctional chelators capable of chelating most nuclides, as well as the development and preparation of bifunctional chelators having higher chelator-to-antibody ratios (DAR values), is an important and a central point of research by those skilled in the art.

Disclosure of Invention

The invention aims to solve the technical problem of preparing a bifunctional macrocyclic chelant derivative with higher DAR value coupling.

In order to solve the technical problem, the invention discloses an NCS-radical-functionalized bi-functional macrocyclic chelant derivative, which has a structure shown in a formula (II):

wherein n is an integer of 1 to 10;

wherein preferably, n is 1, and when n is 1, the structural formula of the compound of formula (II) is as follows:

the compound of the formula (II) is subjected to NCS (NCS) radicalization, so that the technical effects of improving the mildness of coupling conditions, simplifying the coupling process and improving the coupling efficiency can be realized in the coupling process of the bifunctional macrocyclic chelant derivative and the small molecule or macromolecular medicament with amino.

Further, the invention also discloses a preparation method of the NCS group-containing bifunctional macrocyclic chelant derivative, and the preparation method of the NCS group-containing bifunctional macrocyclic chelant derivative compound in the formula (II) comprises the following steps:

in the first step, the first step is to provide,

in the second step, the second step is carried out,

further preferably, the method further comprises a step of purifying the compound of formula (I), wherein the compound of formula (III) is obtained by the first step of reaction, and then the compound of formula (III) is converted into the compound of formula (I), thereby purifying the compound of formula (I), wherein the compound of formula (III) has the following structural formula:

further preferably, in the present invention also specifically disclosed is a process for purifying a compound of formula (I), comprising the steps of:

step A: the compound of the formula (I) reacts with thionyl chloride in methanol solution to prepare the compound of the formula (III),

and (B) step (B): the compound of the formula (III) is hydrolyzed by LiOH in tetrahydrofuran and mixed aqueous solution to obtain the compound of the formula (I),

further, the invention also discloses a method for preparing the compound of the formula (II) by reacting the compound of the formula (I) with thiocarbonyl dichloro,

the invention utilizes the modification of NCS group to improve DAR value of the double-function macrocyclic chelating agent coupled with target molecule, and has mild coupling condition with amino small molecule or macromolecular drug, simple process and high coupling efficiency. Meanwhile, the invention provides a preparation method of the NCS group-containing bifunctional macrocyclic chelant derivative compound D, which is simple in process and suitable for industrial production, and can rapidly and efficiently realize the purification and refining of the compound B through the conversion connection of the purified intermediate compound C.

Drawings

FIG. 1 is a diagram of Compound D ¹ HNMR spectra.

Detailed Description

For a better understanding of the present invention, we will further describe the present invention with reference to specific examples.

EXAMPLE 1 preparation of Compound B

In particular, in this example, et was added to a solution of Compound A (1.00 g,1.30mmol,1.00 eq) in DMF (60.0 mL) ₃ N (1.97 g,19.5mmol,2.71mL,15.0 eq) and HBTU (492 mg,1.30mmol,1.00 eq) in DMF (30.0 mL) was added dropwise to the solution mixture at 20deg.C. The reaction mixture was then stirred at 20℃for 14h. The reaction mixture was concentrated in vacuo to give a residue, which was acidified to pH 5 with 1N HCl, and then concentrated to give a pale residue. The residue was washed with 100ml ACN and then filtered to give compound B (930 mg, crude, 6 HCl) as a yellow solid.

LCMS:[M+H] ⁺ ＝875.5。

EXAMPLE 2 purification of Compound B

The purification of compound B was performed using compound C as a purification intermediate, and in this example, specifically, two steps were included:

step A:

and (B) step (B):

and further, in this embodiment we also disclose that step a and step B are specifically as follows:

step A: a solution of crude compound B (940 mg, 859. Mu. Mol,1.00eq,6 HCl) in MeOH (60.0 mL) was cooled to-5℃and then SOCl was added dropwise ₂ (20.6 g,173mmol,12.5mL,201 eq) and then the reaction mixture was stirred at 25℃for 1h and heated at 45℃for 12h. The reaction mixture was concentrated in vacuo to give a residue and dissolved in water (5.00 ml) with NaHCO ₃ Basification to precipitate a white solid, which was extracted with ethyl acetate (20.0 ml x 3), the organic phase was separated and concentrated in vacuo to one residue. The crude product was purified by preparative HPLC (water+0.225% fa in acetonitrile, 10 min) and lyophilized to give compound C (0.120 g,116 μmol, 25% yield, 89.0% purity) as a white solid.

And (B) step (B): to compound C (200 mg, 218. Mu. Mol,1.00 eq) in THF (10.0 mL) and H ₂ A single portion of LiOH.H was added to a solution of O (10.0 mL) ₂ O (183mg, 4.36mmol,20.0 eq) and then the mixture was stirred at 25℃for 14 hours. THF was removed and the residue was acidified to pH 4 with 1N HCl. The acidified aqueous solution was purified by preparative HPLC (B%: ACN solution in 3% -23% water+0.05% HCl,6 min) to give compound B (126 mg, 112.9. Mu. Mol, 51.8% yield, 96.5% purity, 6 HCl) as a white solid.

LCMS:[M+H] ⁺ ＝875.5。

EXAMPLE 3 preparation of Compound D

In this example, further disclosed is the reaction of compound B with thiocarbonyldichloro to give compound D, which is synthesized as follows:

a specific preparation was carried out by adding thiocarbonyldichloro (105 mg, 914. Mu. Mol, 70.1. Mu.L, 10.0 eq) to a solution of compound B (100 mg, 91.4. Mu. Mol,1.00eq,6 HCl) in deionized water (5.00 mL) and DCM (3 mL) and then stirring the reaction mixture at 15℃for 1 hour. The reaction mixture was extracted with dichloromethane (5.00 ml. Times.3) to remove excess thiocarbonyldichloro and the aqueous layer was lyophilized directly. Yellow solid D (100 mg, 86.1. Mu. Mol,94.2% yield, 97.8% purity, 6 HCl) was obtained.

LCMS：[M+H] ⁺ ＝917.4。

Example 4

By using ¹ HNMR was performed on compound D obtained in example 3, and the detection results are shown in fig. 1.

¹ HNMR(400MHz,D ₂ O)δppm:7.92-7.88(m,2H),7.49-7.45(m,3H),7.36-7.34(m,1H),7.28-7.22(m,4H),7.16(d,J＝8.4Hz,2H),6.98(d,J＝8.4Hz,2H),4.70-4.43(m,16H),4.18(s,2H),4.03(s,2H),3.89-3.87(m,6H),2.20(s,6H)。

Example 5 coupling alignment

5.1 coupling of Compound A with antibodies

The steps are as follows:

50mg of Compound A was placed in a penicillin bottle, 2ml of Tris buffer (pH 5,0.2M) was added, and 10mg of EDC and 10mg of NHS were further added to react at room temperature for 1 hour. 20mg of trastuzumab was added to the penicillin bottle, and the pH of the reaction solution was adjusted to 7.5 using 1M sodium carbonate solution, and the reaction was carried out at room temperature for 2 hours. The liquid was purified using a PD-10 column. Obtaining a conjugate E of the compound A and trastuzumab. The chelator-to-antibody ratio (DAR value) of conjugate a-trastuzumab was determined to be 1.56 by LC-MS.

5.2 conjugation of Compound D to antibodies

The steps are as follows:

placing 0.5mg of compound D into a penicillin bottle, adding 2ml of HEPES sodium salt solution (pH 9,0.2M), adding 20mg of trastuzumab into the penicillin bottle, and reacting for 1h at 37 ℃; and purifying the liquid by using a PD-10 column to obtain a conjugate F of the compound D and the trastuzumab. The DAR value for conjugate D-trastuzumab was determined by LC-MS to be 2.52.

It can be seen that the compound D disclosed by the invention has simpler coupling process, milder condition and higher DAR value compared with the compound A before modification.

What has been described above is a specific embodiment of the present invention. It should be noted that modifications and adaptations to the invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims (3)

1. A process for the preparation of a bifunctional macrocyclic chelant derivative, characterized in that the bifunctional macrocyclic chelant derivative has the structure:

the preparation method comprises the following steps:

in the first step, the first step is to provide,

in the second step, the compound of formula (I) is reacted with thiocarbonyl dichloride to prepare the compound of formula (II),

to deionized water and DCM solution of compound I in the form of 6HCl in an amount of 100mg and deionized water and DCM in an amount of 5.00mL and 3mL, respectively, was added, and the reaction mixture was stirred at 15℃for 1 hour; the reaction mixture was extracted three times with 5.00ml of dichloromethane each to remove excess thiocarbonyl dichloro, and the aqueous layer was lyophilized directly; this gave 100mg of Compound II as a yellow solid in 94.2% yield and 97.8% purity.

2. The method of manufacturing according to claim 1, characterized in that: the method further comprises a step of purifying the compound of the formula (I), wherein the obtained compound of the formula (I) is firstly converted into a compound of the formula (III) in the first step, and then the compound of the formula (III) is converted into the compound of the formula (I), so that the purification of the compound of the formula (I) is completed, wherein the structural formula of the compound of the formula (III) is as follows:

3. the method of manufacturing according to claim 1, characterized in that: the purification method of the compound of formula (I) comprises the following steps:

step A: the compound of the formula (I) reacts with thionyl chloride in methanol solution to prepare the compound of the formula (III),

and (B) step (B): the compound of the formula (III) is hydrolyzed by LiOH in a mixed solution of tetrahydrofuran and water to obtain the compound of the formula (I),